The present invention relates generally to high resolution microscopy techniques. More particularly, the invention provides methods and systems for improved high resolution scanning using apertureless near field scanning optical microscopes (“ANSOM”) that image one or more fluorescent samples with single photon excitation, which we call fluorescence ANSOM (“FANSOM”). But it would be recognized that the invention has a much broader range of applicability. For example, the invention can be applied with other types of images such as Raman scattering, and other multiphoton processes. Additionally, the samples can range from a variety of different fields such as electronics, semiconductor, organic chemistry, life sciences, biotechnology, micro and nanomachining and micro and nanodevices, molecular and biological circuits, and others.
Over the years, significant development of different types of microscopy has occurred. As merely an example, visible light optical microscopy using far field optics including lenses and light evolved from a simple compound microscope that is capable of resolving sizes of about 200 nanometers and greater. Examples of samples that are capable of being viewed using far field optics include biological cells and tissues, and others, which are often, bulk in nature. The resolving ability of such far field optical microscopy is generally limited by the diffraction of light. The diffraction limit for optical resolution has been stretched somewhat for far field imaging of very specific samples to perhaps 150 nanometers using confocal microscopes and other, related, approaches. Accordingly, atomic force microscopes (“AFM”) and scanning optical microscopes including near field scanning optical microscopes were developed. The AFM and near field scanning optical microscopy (“ANSOM”) have been developed to overcome certain limitations of far field optics. The AFM and near field scanning microscopes have also found many applications in biology, chemistry, physics, and materials science.
Near field scanning optical microscopy allows one to take optical images with resolutions below the diffraction limit of light. More particularly, light propagating through a waveguide is forced through a subwavelength aperture, which is then scanned in close proximity to a sample. Such subwavelength aperture techniques create other limitations. Here, physical limitations relate to a skin depth of the metal used to coat the waveguide and various scanning artifacts, which yield resolutions of 30 to 50 nanometers, most typically 50 to 100 nanometers. Apertureless near field scanning microscopes have been proposed and demonstrated to overcome these limitations, among others. Conventional ANSOM often involves using an oscillating sharp probe, which is scanned over the sample. The probe perturbs an incident laser beam, by introducing phase shifts in an electric field or by a periodic occlusion of the sample. Detection techniques are generally used to discriminate light scattered by near field interactions from a far field contribution. Limitations also exist with such ANSOM techniques. Such limitations include contaminated images based upon certain artifacts of the sample topography, and may include others.
A pioneering approach for achieving high resolution spectroscopic information using a scanning microscope is described in U.S. Pat. No. 6,002,471, assigned to California Institute of Technology, Pasadena, Calif., and in the name of Stephen R. Quake (“Quake”). Quake generally provides a system and method for obtaining high resolution spectroscopic information. The system generally includes a support and first optical elements for directing an optical beam at a sample, which is on the support. An optical element for collecting light emitted from the sample to reduce a background noise is also included. Other elements include a spectral dissociating apparatus, a probe, and a probe detection apparatus coupled to the probe. As merely an example, the probe enhances the light level emitted from the sample in the vicinity of the probe. Because this occurs only when the probe is in the immediate vicinity of the sample, detection of this modulation results in very high spatial resolution and chemical detection sensitivity. Fluorescence ANSOM, called FANSOM, has also been demonstrated. Conventional FANSOM often uses a principle of a two photon excitation and electric field enhancement near a tip of the probe. See, T. J. Yang, Guillaume A. Lessard, and Stephen R. Quake, An apertureless near field microscope for fluorescence imaging, Applied Physics Letters, Volume 76, Number 3, Jan. 17, 2000 (“Yang, et al.”). Yang, et al. reports certain results achieved using the FANSOM designed to image fluorescent samples with single photon excitation. FANSOM has demonstrated resolutions in the 10-20 nm range. Although FANSOM appears to be promising, certain practical limitations may still exist.
From the above, it is seen that improved high resolution scanning techniques are desired.